As intracellular parasites (e.g., viruses and Chlamydia) require living cells in order to replicate, diagnosis of infection due to these organisms relies upon the use of either animals (e.g., suckling mice), embryonated eggs, or cell cultures. As cell cultures are much less expensive and are easier to work with than animals or embryonated eggs, cell cultures have long been the mainstay of diagnosis methods for intracellular parasites and viruses in particular. Indeed, cell cultures are the foundation upon which a virology laboratory is built. These cultures may be produced in house from animal tissues or organs, or more commonly, purchased from commercial suppliers.
Regardless of their sources, cell cultures must be maintained over time, in order to ensure a ready supply of cells for growth and diagnosis of infections caused by intracellular parasites. In the laboratory, mammalian cells are routinely frozen in order to minimize the opportunity for contamination of the cultures, guard against handling errors that could result in the loss of the culture, and minimize the number of cell lines that must be handled on a daily basis. Frozen cell culture stocks are also useful for minimizing genetic drift and shift, senescence, and undesirable phenotypic changes that can occur when continuous and finite cell lines are cultured for long time periods.
Freezing methods have been developed to minimize the impact of osmotic shock and intracellular ice crystal formation, two factors that contribute to the loss of cell viability during freezing. Cryoprotectants such as glycerol and dimethylsulfoxide (DMSO) are commonly used to help prevent cell death during freezing. In addition to the use of cryoprotectants, traditional methods use slow cooling (approximately 1° C. per minute) until the cells reach a temperature of −25° C. Once this temperature is attained, the cells can be rapidly cooled to −70° C. or −196° C. (i.e., liquid nitrogen temperature), without further loss of cell viability. Omitting the cryoprotectant or rapid freezing causes the formation of intracellular ice crystals which can rupture cell membranes and result in cell death. By slowly cooling the cells, the external medium becomes supercooled and ice crystal nuclei form in the extracellular fluid. This results in an extracellular environment that contains an artificially elevated salt gradient which, in turn, causes an osmotic gradient. This gradient causes water to diffuse out of the cells and the nonelectrolyte cryoprotectants to diffuse into the cells. This “dehydration” of the cells tends to minimize osmotic shock and intracellular ice crystal formation. (See e.g., Wiedbrauk and Johnston, “Mammalian Cell Culture Procedures, in Manual of Clinical Virology, pages 33-44 [Raven Press, New York, 1993], for a description of these events).
However, commonly used freezing methods require specialized equipment and training. In addition, hazardous chemicals such as DMSO are typically used. Furthermore, thawing of frozen cells maintained in liquid nitrogen poses risks such as explosion of the vials or tubes as well as the danger of loss of cell viability due to improper handling (e.g., slow, rather than rapid thawing). Once the cells have been thawed, the freezing medium must be removed and rinsed from the cells and the culture revived prior to use for growth and/or detection of intracellular parasites. Once revived, the cultures are often placed into formats suitable for the detection and identification of viruses, including multiwell plates (e.g., microtiter plates), tubes, and slides). Thus, the cultures must be transferred from their growth flask to these other formats prior to their use. This necessitates additional equipment and personnel time, prior to the use of the cultures as desired. What is needed are cell cultures and methods that are easy to use, readily available, particularly in ready-to-use formats, require little operator time and/or experience to use, and are reliable.